Excluded volume approximation to protein-solvent interaction. The solvent contact model.

Important properties of globular proteins, such as the stability of its folded state, depend sensitively on interactions with solvent molecules. Existing methods for estimating these interactions, such as the geometrical surface model, are either physically misleading or too time consuming to be applied routinely in energy calculations. As an alternative, we derive here a simple model for the interactions between protein atoms and solvent atoms in the first hydration layer, the solvent contact model, based on the conservation of the total number of atomic contacts, a consequence of the excluded-volume effect. The model has the conceptual advantage that protein-protein contacts and protein-solvent contacts are treated in the same language and the technical advantage that the solvent term becomes a particularly simple function of interatomic distances. The model allows rapid calculation of any physical property that depends only on the number and type of protein-solvent nearest-neighbor contacts. We propose use of the method in the calculation of protein solvation energies, conformational energy calculations, and molecular dynamics simulations.     

Crowding in extremophiles: linkage between solvation and weak protein-protein interactions, stability and dynamics, provides insight into molecular adaptation

The study of the molecular adaptation of microorganisms to extreme environments (solvent, temperature, etc.) has provided tools to investigate the complex relationships between protein-solvent and protein-protein interactions, protein stability and protein dynamics, and how they are modulated by the crowded environment of the cell. We have evaluated protein-solvent and protein-protein interactions by solution experiments (analytical ultracentrifugation, small angle neutron and X-ray scattering, density) and crystallography, and protein dynamics by energy resolved neutron scattering. This review concerns work from our laboratory on (i) proteins from extreme halophilic Archaea, and (ii) psychrophile, mesophile, thermophile and hyperthermophile bacterial cells.     

Theory of cooperative transitions in protein molecules. I. Why denaturation of globular protein is a first-order phase transition

A theory of equilibrium denaturation of proteins is suggested. According to this theory, a cornerstone of protein denaturation is disruption of tight packing of side chains in protein core. Investigation of this disruption is the object of this paper. It is shown that this disruption is an "all-or-none" transition (independent of how compact is the denatured state of a protein and independent of the protein-solvent interactions) because expansion of a globule must exceed some threshold to release rotational isomerization of side chains. Smaller expansion cannot produce entropy compensation of nonbonded energy loss; this is the origin of a free-energy barrier (transition state) between the native and denatured states. The density of the transition state is so high that the solvent cannot penetrate into protein in this state. The results obtained in this paper make it possible to present in the following paper a general phase diagram of protein molecule in solution.     

Amyloid Fibrillation of Insulin under Water-Limited Conditions

Amyloid fibrillation in water-organic mixtures has been widely studied to understand the effect of protein-solvent interactions on the fibrillation process. In this study, we monitored insulin fibrillation in formamide and its methyl derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) in the presence and absence of water. These model solvent systems mimic the cellular environment by providing denaturing conditions and a hydrophobic environment with limited water content. Thioflavin T (ThT) assay revealed that binary mixtures of water with formamide and its methyl derivatives enhanced fibrillation rates and β-sheet abundance, whereas organic solvents suppressed insulin fibrillation. We utilized solution small-angle x-ray scattering (SAXS) and differential scanning calorimetry (DSC) to investigate the correlation between protein-solvent interactions and insulin fibrillation. SAXS experiments combined with simulated annealing of the protein indicated that the degree of denaturation of the hydrophobic core region at residues B11–B17 determines the fibrillation rate. In addition, DSC experiments suggested a crucial role of hydrophobic interactions in the fibrillation process. These results imply that an environment with limited water, which imitates a lipid membrane system, accelerates protein denaturation and the formation of intermolecular hydrophobic interactions during amyloid fibrillation.     

Comparative molecular dynamics of mesophilic and psychrophilic protein homologues studied by 1.2 ns simulations

It is well established that the dynamic motion of proteins plays an important functional role, and that the adaptation of a protein molecule to its environment requires optimization of internal non-covalent interactions and protein-solvent interactions. Serine proteinases in general, and trypsin in particular has been used as a model system in exploring possible structural features for cold adaptation. In this study, a 500 p.s. and a 1200 p.s. molecular dynamics (MD) simulation at 300 K of both anionic salmon trypsin and cationic bovine trypsin are analyzed in terms of molecular flexibility, internal non-covalent interactions and protein-solvent interactions. The present MD simulations do not indicate any increased flexibility of the cold adapted enzyme on an overall basis. However, the apparent higher flexibility and deformability of the active site of anionic salmon trypsin may lower the activation energy for ligand binding and for catalysis, and might be a reason for the increased binding affinity and catalytic efficiency compared to cationic bovine trypsin.     

Amyloid Fibrillation of Insulin under Water-Limited Conditions

Amyloid fibrillation in water-organic mixtures has been widely studied to understand the effect of protein-solvent interactions on the fibrillation process. In this study, we monitored insulin fibrillation in formamide and its methyl derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) in the presence and absence of water. These model solvent systems mimic the cellular environment by providing denaturing conditions and a hydrophobic environment with limited water content. Thioflavin T (ThT) assay revealed that binary mixtures of water with formamide and its methyl derivatives enhanced fibrillation rates and β-sheet abundance, whereas organic solvents suppressed insulin fibrillation. We utilized solution small-angle x-ray scattering (SAXS) and differential scanning calorimetry (DSC) to investigate the correlation between protein-solvent interactions and insulin fibrillation. SAXS experiments combined with simulated annealing of the protein indicated that the degree of denaturation of the hydrophobic core region at residues B11–B17 determines the fibrillation rate. In addition, DSC experiments suggested a crucial role of hydrophobic interactions in the fibrillation process. These results imply that an environment with limited water, which imitates a lipid membrane system, accelerates protein denaturation and the formation of intermolecular hydrophobic interactions during amyloid fibrillation.     

Evaluation of the influence of the internal aqueous solvent structure on electrostatic interactions at the protein-solvent interface by nonlocal continuum electrostatic approach

The dielectric properties of the polar solvent on the protein-solvent interface at small intercharge distances are still poorly explored. To deconvolute this problem and to evaluate the pair-wise electrostatic interaction (PEI) energies of the point charges located at the protein-solvent interface we used a nonlocal (NL) electrostatic approach along with a static NL dielectric response function of water. The influence of the aqueous solvent microstructure (determined by a strong nonelectrostatic correlation effect between water dipoles within the orientational Debye polarization mode) on electrostatic interactions at the interface was studied in our work. It was shown that the PEI energies can be significantly higher than the energies evaluated by the classical (local) consideration, treating water molecules as belonging to the bulk solvent with a high dielectric constant. Our analysis points to the existence of a rather extended, effective low-dielectric interfacial water shell on the protein surface. The main dielectric properties of this shell (effective thickness together with distance- and orientation-dependent dielectric permittivity function) were evaluated. The dramatic role of this shell was demonstrated when estimating the protein association rate constants.     

Imaging the migration pathways for O2, CO, NO, and Xe inside myoglobin

Myoglobin (Mb) is perhaps the most studied protein, experimentally and theoretically. Despite the wealth of known details regarding the gas migration processes inside Mb, there exists no fully conclusive picture of these pathways. We address this deficiency by presenting a complete map of all the gas migration pathways inside Mb for small gas ligands (O2, NO, CO, and Xe). To accomplish this, we introduce a computational approach for studying gas migration, which we call implicit ligand sampling. Rather than simulating actual gas migration events, we infer the location of gas migration pathways based on a free-energy perturbation approach applied to simulations of Mb's dynamical fluctuations at equilibrium in the absence of ligand. The method provides complete three-dimensional maps of the potential of mean force of gas ligand placement anywhere inside a protein-solvent system. From such free-energy maps we identify each gas docking site, the pathways between these sites, to the heme and to the external solution. Our maps match previously known features of these pathways in Mb, but also point to the existence of additional exits from the protein matrix in regions that are not easily probed by experiment. We also compare the pathway maps of Mb for different gas ligands and for different animal species.     

Halophilic adaptation: novel solvent protein interactions observed in the 2.9 and 2.6 A resolution structures of the wild type and a mutant of malate dehydrogenase from Haloarcula marismortui

Previous biophysical studies of tetrameric malate dehydrogenase from the halophilic archaeon Haloarcula marismortui (Hm MalDH) have revealed the importance of protein-solvent interactions for its adaptation to molar salt conditions that strongly affect protein solubility, stability, and activity, in general. The structures of the E267R stability mutant of apo (-NADH) Hm MalDH determined to 2.6 A resolution and of apo (-NADH) wild type Hm MalDH determined to 2.9 A resolution, presented here, highlight a variety of novel protein-solvent features involved in halophilic adaptation. The tetramer appears to be stabilized by ordered water molecule networks and intersubunit complex salt bridges "locked" in by bound solvent chloride and sodium ions. The E267R mutation points into a central ordered water cavity, disrupting protein-solvent interactions. The analysis of the crystal structures showed that halophilic adaptation is not aimed uniquely at "protecting" the enzyme from the extreme salt conditions, as may have been expected, but, on the contrary, consists of mechanisms that harness the high ionic concentration in the environment.     

Calculation of the electric potential in the active site cleft due to alpha-helix dipoles

A macroscopic dielectric model has been used to set up the electrostatic equation for the protein-solvent system. A numerical method of solution has been applied, enabling calculation of the electric potential outside a protein due to the charges within the protein. The glycolytic enzyme phosphoglycerate mutase, which is an α/β protein binding negatively charged substrates, has been studied. Modelling the helix dipoles with positive and negative charges shows that the α-helical structure could stabilize negatively charged substrates in the active site cleft of an enzyme with an energy of a few kT.     

A computational method for design of connected catalytic networks in proteins

Computational design of new active sites has generally proceeded by geometrically defining interactions between the reaction transition state(s) and surrounding side‐chain functional groups which maximize transition‐state stabilization, and then searching for sites in protein scaffolds where the specified side‐chain–transition‐state interactions can be realized. A limitation of this approach is that the interactions between the side chains themselves are not constrained. An extensive connected hydrogen bond network involving the catalytic residues was observed in a designed retroaldolase following directed evolution. Such connected networks could increase catalytic activity by preorganizing active site residues in catalytically competent orientations, and enabling concerted interactions between side chains during catalysis, for example, proton shuffling. We developed a method for designing active sites in which the catalytic side chains, in addition to making interactions with the transition state, are also involved in extensive hydrogen bond networks. Because of the added constraint of hydrogen‐bond connectivity between the catalytic side chains, to find solutions, a wider range of interactions between these side chains and the transition state must be considered. Our new method starts from a ChemDraw‐like two‐dimensional representation of the transition state with hydrogen‐bond donors, acceptors, and covalent interaction sites indicated, and all placements of side‐chain functional groups that make the indicated interactions with the transition state, and are fully connected in a single hydrogen‐bond network are systematically enumerated. The RosettaMatch method can then be used to identify realizations of these fully‐connected active sites in protein scaffolds. The method generates many fully‐connected active site solutions for a set of model reactions that are promising starting points for the design of fully‐preorganized enzyme catalysts.     

Influence of the solvent structure on the electrostatic interactions in proteins

The proper estimation of the influence of the many-body dynamic solvent microstructure on a pairwise electrostatic interaction (PEI) at the protein-solvent interface is very important for solving many biophysical problems. In this work, the PEI energy was calculated for a system that models the interface between a protein and an aqueous solvent. The concept of nonlocal electrostatics for interfacial electrochemical systems was used to evaluate the contribution of a solvent orientational polarization, correlated by the network of hydrogen bonds, into the PEI energy in proteins. The analytical expression for this energy was obtained in the form of Coulomb's law with an effective distance-dependent dielectric function. The asymptotic and numerical analysis carried out for this function revealed several features of dielectric heterogeneity at the protein-solvent interface. For charges located in close proximity to this interface, the values of the dielectric function for the short-distance electrostatic interactions were found to be remarkably smaller than those determined by the classical model, in which the solvent was considered as the uniform dielectric medium of high dielectric constant. Our results have shown that taking into consideration the dynamic solvent microstructure remarkably increases the value of the PEI energy at the protein-solvent interface.     

How to guarantee optimal stability for most representative structures in the Protein Data Bank

We proposed recently an optimization method to derive energy parameters for simplified models of protein folding. The method is based on the maximization of the thermodynamic average of the overlap between protein native structures and a Boltzmann ensemble of alternative structures. Such a condition enforces protein models whose ground states are most similar to the corresponding native states. We present here an extensive testing of the method for a simple residue-residue contact energy function and for alternative structures generated by threading. The optimized energy function guarantees high stability and a well-correlated energy landscape to most representative structures in the PDB database. Failures in the recognition of the native structure can be attributed to the neglect of interactions between different chains in oligomeric proteins or with cofactors. When these are taken into account, only very few X-ray structures are not recognized. Most of them are short inhibitors or fragments and one is a structure that presents serious inconsistencies. Finally, we discuss the reasons that make NMR structures more difficult to recognizeCopyright 2001 Wiley-Liss, Inc.     

Backbone-driven collapse in unfolded protein chains

Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.     

Side chain-backbone hydrogen bonding contributes to helix stability in peptides derived from an alpha-helical region of carboxypeptidase A

Recently, Presta and Rose proposed that a necessary condition for helix formation is the presence of residues at the N- and C-termini (called NTBs and CTBs) whose side chains can form hydrogen bonds with the initial four amides and the last four carbonyls of the helix, which otherwise lack intrahelical hydrogen bonding partners. We have tested this hypothesis by conformational analysis by circular dichroism (CD) of a synthetic peptide corresponding to a region (171-188) of the protein carboxypeptidase A; in the protein, residues 174 to 186 are helical and are flanked by NTBs and CTBs. Since helix formation in this peptide may also be stabilized by electrostatic interactions, we have compared the helical content of the native peptide with that of several modified peptides designed to enable dissection of different contributions to helix stability. As expected, helix dipole interactions appear to contribute substantially, but we conclude that hydrogen bonding interactions as proposed by Presta and Rose also stabilize helix formation. To assist in comparison of different peptides, we have introduced two concentration-independent CD parameters which are sensitive probes of helix formation.     

Four-body contact potentials derived from two protein datasets to discriminate native structures from decoys

Two-body inter-residue contact potentials for proteins have often been extracted and extensively used for threading. Here, we have developed a new scheme to derive four-body contact potentials as a way to consider protein interactions in a more cooperative model. We use several datasets of protein native structures to demonstrate that around 500 chains are sufficient to provide a good estimate of these four-body contact potentials by obtaining convergent threading results. We also have deliberately chosen two sets of protein native structures differing in resolution, one with all chains' resolution better than 1.5 A and the other with 94.2% of the structures having a resolution worse than 1.5 A to investigate whether potentials from well-refined protein datasets perform better in threading. However, potentials from well-refined proteins did not generate statistically significant better threading results. Our four-body contact potentials can discriminate well between native structures and partially unfolded or deliberately misfolded structures. Compared with another set of four-body contact potentials derived by using a Delaunay tessellation algorithm, our four-body contact potentials appear to offer a better characterization of the interactions between backbones and side chains and provide better threading results, somewhat complementary to those found using other potentials.     

Hydrophile scanning as a complement to alanine scanning for exploring and manipulating protein-protein recognition: application to the Bim BH3 domain

Alanine scanning has been widely employed as a method of identifying side chains that play important roles in protein-protein and protein-peptide interactions. Here we show how an analogous and complementary technique, hydrophile scanning, can provide additional insight on such interactions. Mutation of a wild-type residue to alanine removes most of the side-chain atoms, and the effect of this removal is typically interpreted to indicate contribution of the deleted side chain to the stability of the complex. Hydrophile scanning involves systematic mutation of wild-type residues to a cationic or anionic residue (lysine or glutamic acid, in this case). We find that the results of these mutations provide insights on interactions between polypeptide surfaces that are complementary to the information obtained via alanine scanning. We have applied this technique to a peptide that corresponds to the BH3 domain of the pro-apoptotic protein Bim. The wild-type Bim BH3 domain binds strongly to the anti-apoptotic proteins Bcl-x(L) and Mcl-1. Combining information from the alanine, lysine, and glutamic acid scans has enabled us to identify Bim BH3 domain mutants containing only two or three sequence changes that bind very selectively either to Bcl-x(L) or Mcl-1. Our findings suggest that hydrophile scanning may prove to be a broadly useful tool for revealing sources of protein-protein recognition and for engineering selectivity into natural sequences.     

Backbone and side-chain contributions in protein denaturation by urea

Urea is a commonly used protein denaturant, and it is of great interest to determine its interaction with various protein groups to elucidate the molecular basis of its effect on protein stability. Using the Trp-cage miniprotein as a model system, we report what we believe to be the first computation of changes in the preferential interaction coefficient of the protein upon urea denaturation from molecular-dynamics simulations and examine the contributions from the backbone and the side-chain groups. The preferential interaction is obtained from reversible folding/unfolding replica exchange molecular-dynamics simulations of Trp-cage in presence of urea, over a wide range of urea concentration. The increase in preferential interaction upon unfolding is dominated by the side-chain contribution, rather than the backbone. Similar trends are observed in simulations using two different force fields, Amber94 and Amber99sb, for the protein. The magnitudes of the side-chain and backbone contributions differ in the two force fields, despite containing identical protein-solvent interaction terms. The differences arise from the unfolded ensembles sampled, with Amber99sb favoring conformations with larger surface area and lower helical content. These results emphasize the importance of the side-chain interactions with urea in protein denaturation, and highlight the dependence of the computed driving forces on the unfolded ensemble sampled.     

Probing protein structure and dynamics by second-derivative ultraviolet absorption analysis of cation-{pi} interactions

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation-pi interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second-derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N-acetylated C-ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation-pi interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.